The present invention relates to a protein-immobilizing solid phase, a polynucleotide-immobilizing solid phase, and a nucleic acid recovery method.
New functional proteins are expected to contribute to various applications in the field of biotechnology, such as in pharmaceuticals, detergents, food processing, reagents for research and development, clinical analyses as well as bioenergy and biosensors.
Although protein engineering techniques, consisting of using human intellect to design proteins based on protein structural information, have been primarily used when acquiring new functional proteins, since screening methods more efficient than those used in the past are required to acquire more useful proteins, expectations are being placed on molecular evolutionary engineering techniques that consist of randomly repeating modification and screening of protein molecular structure.
The cDNA display method, which is a type of molecular evolutionary engineering technique, is a method for associating genotype and phenotype, and consists of the use of a nucleic acid linker to link a protein (phenotype) with mRNA encoding the protein and reverse-transcribed cDNA (genotype). Since the mRNA/cDNA-protein linkage structure is extremely stable, screening can be carried out in various environments by using this nucleic acid linker.
The cDNA display method is characterized by the presence of puromycin in a nucleic acid linker that links a protein with a polynucleotide that encodes that protein (see Japanese Patent No. 4318721).
Puromycin is a protein synthesis inhibitor having a structure that resembles the 3′-terminal of aminoacyl-tRNA, and under prescribed conditions, specifically covalently bonds to the C-terminal of protein during elongation on a ribosome.
Methods for screening useful proteins using the cDNA display method consist of the series of steps described below.
First, a nucleic acid linker containing puromycin is coupled to mRNA, protein is synthesized from the mRNA using a cell-free translation system, and the synthesized protein and mRNA encoding that protein are linked through puromycin to form a complex (mRNA-nucleic acid linker-protein complex) (see Nemoto, et al., FEBS Lett., Vol. 414, pp. 405-408, 1997).
Next, a library of this mRNA-nucleic acid linker-protein complex is prepared, the prepared mRNA-nucleic acid linker-protein complex is reverse-transcribed with reverse transcriptase to synthesize cDNA, and this synthesized cDNA is used to prepare an mRNA/cDNA-nucleic acid linker-protein complex library, followed by selecting a protein having a desired function. The protein can be identified by analyzing the base sequence of the cDNA in the selected mRNA/cDNA-nucleic acid linker-protein complex. Reverse transcription may also be carried out prior to protein selection (see Yamaguchi, et al., Nucleic Acids Res., Vol. 37, p. e108, 2009).
A protein array, in which a library of the aforementioned mRNA (or mRNA/cDNA)-nucleic acid linker-protein complex is immobilized on a substrate, is useful as a tool for acquiring functional protein in a short period of time by comprehensive analysis (see Japanese Unexamined Patent Application, First Publication No. 2008-116218).